Summary of Work: The bacterial pathogenesis of nontypeable Haemophilus influenzae (NTHi) otitis media is not well understood. Evidence suggests that bacterial adherence and delivery of bacterial toxins on the target epithelial cells are critical for the pathogenesis of otitis media. The efforts of this project are to immortalize middle ear epithelial cells, to study bacterial adherence on these immortalized cell lines with human otitis media pathogens, to identify bacterial adhesion molecules and their potential binding receptors, and to determine if these molecules may serve as antigens or targets for prevention or therapy. Two middle ear epithelial cell lines from rats and chinchillas and one Eustachian tube epithelial cell line from rats have been immortalized by infecting the primary cell cultures with adeno 12-SV40 hybrid virus. In this past year, we have finished the final process of characterizing these cell lines using the techniques of cell biology and immunochemistry. These cell lines will provide an unlimited source of homogeneous progenitors for the study of the pathogenesis of otitis media. We are in the process of studying the bacterial adherence on the immortalized cell lines with strains collected from middle ear effusion or nasopharynges of persons with otitis media.